DNA sequence regulating plant fruit-specific expression

ABSTRACT

The present invention has for its object to provide a DNA sequence capable of regulating the expression of a desired gene specifically in the fruit of a plant, a plasmid containing that DNA sequence as well as a plant cell, plant body and microorganism transformed with the plasmid. 
     One aspect of the present invention is constituted by a fruit-specific expression-regulating unit DNA sequence which comprises a region comprising the base sequence shown under SEQ ID NO:1.

TECHNICAL FIELD

The present invention relates to a DNA sequence for gene expression andregulation in plant fruits and the use thereof. More particularly, itrelates to a DNA sequence for specifically regulating gene expression inplant fruits, a plasmid containing that DNA sequence, a plant cell,plant body and microorganism transformed with that plasmid.

BACKGROUND OF THE INVENTION

A technique is known for creating a transgenic plant transformed withDNA sequences comprising a gene coding for a desired protein capable ofbeing expressed in a plant and a certain promoter sequence in order toexpress that protein. One of such promoters is the cauliflower mosaicvirus (CaMV) 35S promoter capable of allowing non-tissue-specificexpression of a desired protein in a plant. As regards proteinexpression systems in plants, inducible gene promoters capable ofreacting with an exogenous chemical substance have also been reported,for example a system in which the tetracycline-inducible Tn10 Tetrepressor is utilized (The Plant J. (2), 397, 1992) and a system inwhich the steroid hormone-inducible rat glucocorticoid receptor isutilized (The Plant J. (11), 605, 1997). As regards base sequencesinvolved in tissue-specific expression of plant genes, the so-called RYcore sequence CATGCAT associated with a gene that is expressed in aseed-specific manner is known (Plant Physiol. (98), 387, 1992). It hasalso been disclosed that a desired protein can be expressed in raspberryfruit by using the raspberry drul promoter (Japanese Kohyo Publication2000-503848).

Cucumisin, a plant-derived protease, is a thermostable alkaline serineprotease which is abundantly accumulated in melon juice (Agric. Biol.Chem. (53), 1009, 1989) and cDNA therefor has been cloned (J. Biol.Chem. (52), 32725, 1994)). However, the regulating mechanisms of itsfruit-specific expression remain unknown.

Transgenic crop plants transformed with a herbicide resistance gene orthe like by utilizing the technology of introducing a heterologous DNAinto plants to create transgenic plants have already been utilized infood production. On the other hand, another method of utilizingtransgenic plants, namely the development into the so-called molecularagriculture in which plants are caused to produce useful proteins andthe products are extracted for utilization, cannot yet be said to havebeen established as an industry. When plant cells are or a plant body iscaused to produce a useful protein or a useful substance resulting fromconversion thereof, there arise the possibility that if the product isexpressed in the leaf, stem or root, the extraction efficiency orpurification/recovery of the product may become or encounter problems insome not only in the case of non-tissue-specific expression in a plantbut also in the case of specific expression. A promoter readilycontrollable and ensuring good production efficiency is thus desired.The fruit is a storage organ by far greater in volume than seeds and, inparticular, the gourd family, typically melon, is characterized by itsproducing a great number of relatively large fruits. If a technology ofaccumulating a useful protein or useful substance in those fruits orgiving them desired properties is established, the technology willbecome a very useful one. While there are several reports about the basesequences involved in expression specific to the seed, leaf or root ofplants, there is no report found about the identification andutilization of a base sequence, in particular a melon promoter sequence,involved in fruit-specific expression.

SUMMARY OF THE INVENTION

In view of the above-discussed state of the art, the present inventionhas for its object to provide a DNA sequence capable of regulating theexpression of a desired gene specifically in the fruit of a plant, aplasmid containing that DNA sequence as well as a plant cell, plant bodyand microorganism transformed with the plasmid.

As a result of intensive investigations, the present inventors couldidentify a novel regulatory DNA sequence involved in fruit-specificexpression in a region upstream of the melon-derived cucumisin genecoding for a protein expressed at a high level specifically in the fruitand found that the DNA sequence functions in site-specific expression inthe plant body. Based these findings, they have completed the presentinvention.

Thus, in a first aspect, the present invention consists in afruit-specific expression regulating unit DNA sequence

which comprises a region comprising the base sequence shown under SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7. The DNA sequenceaccording to the first aspect of the present invention can be preferablyregulated by a transcription factor capable of binding to the basesequence shown under SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:7 and involved in fruit-specific expression.

In a second aspect, the present invention relates to a plasmid

which comprises a DNA coding for a structural gene capable of beingexpressed in a plant or for a corresponding antisense RNA, a promoterand a terminator capable of functioning in the plant, and the DNAsequence according to the first aspect of the invention.

In a third aspect, the present invention relates to a plant cell or aplant

which is able to express a protein or a corresponding antisense RNAencoded by a desired structural gene in a fruit-specific manner with theplasmid according to the second aspect of the present.

In a fourth aspect, the present invention relates to a microorganismharboring the plasmid according to the second aspect of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a plasmid, pM1-B2-5, containinga cucumisin promoter region as an insert DNA.

FIG. 2 is a schematic representation of a plasmid, pSKGU3C, for promoteractivity measurement.

FIG. 3 is a schematic representation of the cucumisin promoter regionDNA sequence in a plasmid, pSKGU3C, as inserted at the XbaI-BamHI sitethereof, and of plasmids constructed therefrom.

FIG. 4 is a schematic representation of the DNA sequence inserted into aplasmid, pSKGU3C, at the XbaI-BamHI site thereof in constructing aplasmid, pKGX.

FIG. 5 is a schematic representation of the DNA sequence inserted into aplasmid, pSKGU3C, at the XbaI-BamHI site thereof in constructing aplasmid, pX.

FIG. 6 is a schematic representation of a plasmid, pBI221.

FIG. 7 is a schematic representation of a plasmid, p35S-INT-LUC+.

DETAILED DISCLOSURE OF THE INVENTION

In the following, the present invention is described in detail.

In a first aspect, the present invention is directed to a fruit-specificexpression regulating DNA sequence capable of functioning in plant cellswhich is a DNA sequence being able to function as a fruit-specificexpression regulating unit and comprises a region comprising the basesequence shown under SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:7.

The DNA sequence according to the first aspect of the invention maycontain one of the sequences shown under SEQ ID NO:1, SEQ ID NO:2 andSEQ ID NO:7 singly or may contain two or more of them. As a specificregion containing the sequence shown under SEQ ID NO:1 or SEQ ID NO:7and/or the sequence shown under SEQ ID NO:2, there may be mentioned aregion which is a melon gene and contains the base sequence shown underSEQ ID NO:3.

The DNA sequence according to the first aspect of the invention is asequence containing a region derived from the cucumisin promoter gene ofmelon and can regulate the fruit-specific expression and, therefore,when the DNA sequence of the invention is used, a fruit can be providedwith any desired property. For example, it is possible to produce auseful substance in a site-specific manner in fruits, which arepromising storage organs in plants, or inhibit the transcription of aspecific gene to be expressed in fruit.

The DNA sequence according to the first aspect of the invention ispreferably regulated by a transcription factor involved infruit-specific expression and capable of binding to the base sequenceshown under SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7.

The above-mentioned transcription factor includes, among others, anucleoprotein binding to the DNA sequence shown under SEQ ID NO:7, aGATA box-binding protein, an I-box-binding protein, a G-box-bindingprotein, and a protein capable of binding to the Gb probe in gel shiftassay as shown herein in Example 7.

For the DNA sequence according to the first aspect of the invention tofunction as a fruit-specific expression-regulating promoter, it isnecessary that a transcription factor should bind to the above DNAsequence. Therefore, the expression of a desired useful substance, forinstance, can be regulated by gene transfer of the transcription factorinto cells.

In accordance with the first aspect of the invention, those basesequences derived from the respective base sequences by partialmutation, substitution, deletion or a like procedure that will notimpair the functions of course fall within the scope of the presentinvention.

In accordance with the first aspect of the invention, it is alsopossible to make a protein encoded by a structural gene regulated by apromoter having activity in plants be expressed in a fruit-specificmanner by linking, inserting or substituting a base sequence whichcontained in the sequence shown under SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 or SEQ ID NO:7 to, into or for a promoter capable of functioning inother plants, for example the cauliflower mosaic virus (CaMV) 35Spromoter.

Although any of those so-far known definite plant hormone-responsiveelements, such as the ethylene-responsive element and auxin-responsiveelement, cannot be found in the DNA sequence according to the firstaspect of the invention, the sequence may function in cooperation withan unidentified plant hormone element or the like to cause thefruit-specific expression.

Further, as shown in Table 1 in Example 4, it is suggested that the DNAsequence shown as bases NO.1 to 20 in SEQ ID NO:7 might include a DNAsequence functioning as an expression inhibiting element. It is readilyanticipated that a procedure, such as deletion or substitution, appliedto the DNA sequence in that portion might exert an obvious influence onthe fruit-specific expression.

In the practice of the invention according to the first aspect thereof,the fruit is not particularly restricted but widely includes fruitsobtainable from seed plants. Advantageously used are, however, plants ofthe gourd family (Cucurbitaceae), such as melon, watermelon, gourd,cucumber and cushaw (pumpkin). Melon is preferred among others, since itis great in fruit size, hence can store a large amount of a usefulsubstance.

The base sequences shown under SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:7 are derived from the melon cucumisin promoter sequence, andthe DNA sequence according to the first aspect of the invention can beobtained in the conventional manner, for example, by extracting genomicDNA from melon and carrying out the IPCR using primers prepared based onthe exon sequence of cucumisin.

A regulatory function of the DNA sequence according to the first aspectof the invention in a plant body or plant cells can be checked with asufficient level of reliability by transient expression using theparticle bombardment method for site-specific expression examination.

In accordance with the second aspect, the invention consists in a DNAcoding for a structural gene capable of being expressed in plants or acorresponding antisense RNA as well as a plasmid having a promoter and aterminator capable of functioning in plants and the DNA sequenceaccording to the first aspect of the invention.

The above structural gene and antisense RNA are not particularlyrestricted but can adequately be selected according to the intendedpurpose.

The above-mentioned promoter is not particularly restricted but may beany of those capable of functioning in plant cells. Thus, mention may bemade, for example, of the cauliflower mosaic virus (CaMV) 35S promoter,the octopine synthesis gene promoter of Agrobacterium, the tobacco PR1agene promoter, and the tomato ribulose 1,5-diphosphate carboxylaseoxidase small subunit promoter.

The above-mentioned terminator is not particularly restricted but may beany of those capable of functioning in plant cells. Thus, there may bementioned, for example, the RuBisCO 3C gene or plant gene-derivednopaline synthesis gene terminator, and the garlic virus GV1 or GV2 geneterminator.

The plasmid according to the second aspect of the invention can beconstructed in the conventional manner using one of various knownvectors.

When the plasmid is introduced into cells by the electroporation,particle bombardment, microinjection or fusion method, for instance, theabove vector is not particularly restricted but use can be made of anyof the known vectors, such as pBR322, pBR325, pUC19, pUC119,pBluescript, pBluescript SK and pBluescript II SK. When the plasmidtransfer is to be realized by the method of infecting plant cells withAgrobacterium, mention may be made, for example, of such vectors as thebinary vectors pBI101 (product of Clontech), pBI121 (product ofClontech) and pBI221 (product of Clontech).

The plasmid according to the second aspect of the invention can beobtained by inserting, into such a vector, the DNA sequence according tothe first aspect of the invention, namely a DNA coding for a structuralgene capable of being expressed in plants or a corresponding antisenseRNA, and a promoter and a terminator capable of functioning in plants.

Furthermore, when a promoter having a fruit-specific expressionregulating DNA sequence according to the first aspect of the inventionis used to express of a protein, it is also possible to positively causesecretion a protein in a fruit or fruit juice or positively allowing thematter conversion reaction(s) involving such a protein or a plurality ofsuch proteins to proceed in a plant fruit for the production of a usefulsubstance resulting from such reaction(s) by linking, upstream of theprotein-encoding gene capable of being expressed in plants, a signalsequence necessary for extracellular secretion of the protein, forexample the secretory signal-encoding DNA sequence of the meloncucumisin gene or the secretory signal-encoding DNA sequence of someother gene or, downstream from the gene coding for said protein, a DNAsequence coding for a localization signal governing the localizabilityof the protein in cells.

Furthermore, it is also possible to cause expression of a correspondingantisense RNA of the gene coding for a protein involved in the overripeof fruit, in addition to the expression of the desired protein, for animprovement in storability or like controlling purposes.

For facilitating the transformant plant selection, a selective markergene may be inserted into the plasmid according to the second aspect ofthe invention. The selective marker gene may be, for example, anantibiotic resistance gene, such as the G418, hygromycin, bleomycin,kanamycin, gentamicin, chloramphenicol resistance gene or the like. Whenthe plasmid contains an antibiotic resistance gene, a transgenic plant,namely a plant transformed with the plasmid of the invention introducedtherein, can be selected with ease by selecting a plant capable ofgrowing in a medium containing the antibiotic.

By transforming a plant cell or a plant body using the plasmid accordingto the second aspect of the invention, it becomes possible tospecifically produce a desired useful substance, or inhibit theproduction of an unnecessary substance, in the fruit of that plant.

As the method of introducing the DNA sequence according to the firstaspect of the invention or the plasmid according to the second aspect ofthe invention into a plant cell or a plant body for the purpose offruit-specific expression, there may be such known methods as thetechnique of plant cell infection with Agrobacterium, theelectroporation method, the particle gun method, the microinjectionmethod, and the technique comprising fusion of protoplast with avector-containing small cell, cell or lysosome, for instance.

In accordance with the third aspect, the invention provides a plant cellor plant body capable of fruit-specifically expressing the protein orantisense RNA encoded by the target structural gene owing to theoccurrence of the plasmid according to the second aspect of theinvention.

The above plant cell or plant body is not particularly restricted butmay be any of those capable of producing fruit. Preferred are, however,plants of the gourd family, such as melon, watermelon, gourd, cucumberand cushaw.

In accordance with the fourth aspect, the invention provides amicroorganism harboring the plasmid according to the second aspect ofthe invention.

As the above microorganism, there may be mentioned, among others,Agrobacterium species and Escherichia coli.

It is also possible to produce the plant cell or plant body according tothe third aspect of the invention using an Agrobacterium strainharboring the plasmid according to the second aspect of the invention.

When a plant is infected with such an Agrobacterium strain, part of theplasmid DNA harbored in the bacterial cells is transferred to the plantgenome. Thus, by utilizing this characteristic, it is possible tointroduce, into plants, the DNA according to the first aspect of theinvention or the DNA coding for a structural gene capable of beingexpressed in plants or a corresponding antisense RNA, together with apromoter and a terminator capable of functioning in plants.

In cases where the plasmid according to the second aspect of theinvention has no vir region, the Agrobacterium strain to be used isrequired to have another plasmid containing the vir region.

BEST MODES FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in furtherdetail. They are, however, by no means limitative of the scope of thepresent invention.

EXAMPLE 1 Isolation of the Cucumisin Protein Promoter from Melon GenomicDNA

(1) Preparation of Melon Genomic DNA

Leaves (10 g) of a seedling of muskmelon (Cucumis melo L. reticulatuscv. Teresa) were cut to pieces and ground in liquid nitrogen. The groundmatter was suspended in 2×CTAB buffer (2% CTAB, 1.4 M NaCl, 100 mMTris-HCl (pH 8.0), 20 mM EDTA, 1% polyvinylpyrrolidone) and, after 10minutes of heating at 55° C., chloroform-isoamyl alcohol (24:1) wasadded, and the mixture was gently mixed up by inverting at roomtemperature for 30 minutes. After 15 minutes of centrifugation at14000×g, chloroform-isoamyl alcohol (24:1) was added to the upper layer,and 1×CTAB buffer to the intermediate layer and bottom layer, followedby gentle mixing up by inverting and centrifugation. The upper layerswere combined, and a 1/10 volume of 10% CTAB solution (10% CTAB, 0.7 MNaCl) was added, followed by mixing up by inverting. Further, an equalvolume of CTAB precipitation buffer (1% CTAB, 50 mM Tris-HCl (pH 8.0),10 mM EDTA) was added, followed by gentle mixing up by inverting andcentrifugation. 1 M NaCl-TE buffer (1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1mM EDTA) was added to the precipitate obtained for dissolution of theprecipitate. Thereto was added an equal volume of isopropanol and, aftergentle mixing up by inverting, the mixture was centrifuged. Theprecipitate obtained was washed with 70% ethanol and dried and dissolvedin TE buffer. After dissolution, RNase A and proteinase K were added tothe respective final concentrations of 5 μg/ml and 10 μg/ml and, after30 minutes of heating at 50° C., the mixture was extracted withphenol-chloroform-isoamyl alcohol (25:24:1). A 1/10 volume of 3 M sodiumacetate solution (pH 5.2) and an equal volume of isopropanol were addedto the upper layer, the resulting mixture was centrifuged, and theprecipitate was washed with 70% ethanol and dissolved in TE buffer togive a genomic DNA solution.

(2) Promoter Region Cloning by the IPCR (Inverse PCR)

The thus-prepared DNA solution containing 1 μg of melon genomic DNA wastreated overnight with 10 units each of the restriction enzymes BamHIand BglII at 37° C. The digested DNA fragments were recovered by ethanolprecipitation, and subjected to reaction using 10 units of T4 DNA ligaseat 15° C. for 12 hours. To this DNA fraction were added 10 ng ofcucumisin primer 1 (SEQ ID NO:4) and 10 ng of cucumisin primer 2 (SEQ IDNO:5), and a DNTP mixture (to each final dNTP concentration of 0.2 mM)and 0.5 μl of AmpliTaq Gold DNA Polymerase were added to give a 100-μlsolution, and the PCR was carried out using a Zymoreactor (product ofATTO, type AB-1800); thus, after 10 minutes of treatment at 95° C., 30cycles each comprising 1 minute at 94° C., 2 minutes at 55° C. and 3minutes at 72° C. were repeated, followed by 7 minutes of final PCRtreatment at 72° C. After completion of the reaction, the amplified DNAfragment was inserted into a plasmid fragment prepared by cleavingpBluescript II SK with the restriction enzyme SmaI and adding one base Tto the 5′ terminus thereof, to give a plasmid, pM1-B2-5 (FIG. 1). TheDNA sequencing of the insert fragment was carried out using a DNAsequencer (model 4000L; product of LI-COR, Inc., NE, USA) and theSequiTherm EXCEL II Cycle Sequencing Kit-LC (product of EpicentreTechnologies, Madison, Wis., USA). As a result, the base sequence of anabout 1.2 kb cucumisin promoter site shown under SEQ ID NO:3 wasdetermined.

EXAMPLE 2

When an expression analysis is carried out using the promoter sequenceof an isolated gene, it is desirable to construct a fusion generesulting from linking of a reporter gene enabling an easy activityassay at a site as close as possible to the transcription initiationpoint of the original gene. Therefore, the transcription initiationpoint of the isolated cucumisin gene was determined.

The Ex primer (SEQ ID NO:6) terminally labeled with [γ-³²P]ATP using T4polynucleotide kinase was allowed overnight at 42° C. to hybridize with20 μg of poly(A)+ RNA extracted from the core of an immature muskmelonfruit at 10 days after pollination. Using the reaction mixture, theprimer extension reaction by reverse transcription was carried out byadding 12.5 units of AMV RT (product of LIFE SCIENCE, INC.) andincubating for 1 hour at 55° C. Subsequently, the ethanol precipitationproduct was subjected to polyacrylamide gel electrophoresis with asequencing gel composition. As a result, only one transcriptioninitiation point was found for the cucumisin gene (a: adenine followingSEQ ID NO:3).

EXAMPLE 3 Construction of a Melon Promoter-GUS Fusion Gene

A vector for promoter activity assaying, pSKGUS3C, was constructed byjoining, to the plasmid pBluescript II SK, the pBI221 vector (ClontechLaboratories, Inc.)-derived β-glucuronidase (GUS) gene, as a reportergene, and the RuBisCO 3C gene, as a terminator (FIG. 2). Then, PCR wascarried out using the pM1-B2-5 plasmid as the template, together with aprimer having a restriction enzyme XbaI site as designed to successivelydelete the upstream portion of the cucumisin promoter sequence and aprimer having a BamHI site as resulting from cleavage at a site one baseupstream of the transcription initiation point. The thus-obtained DNAfragments differing in length were inserted between the restrictionenzyme sites XbaI and BamHI occurring upstream of the GUS gene ofpSKGUS3C to construct cucumisin promoter-GUS fusion genes. In indicatingbases in a promoter region, the base immediately before thetranscription initiation point is generally numbered −1. Thus, when the3′ terminus of the base sequence under SEQ ID NO:3 is numbered −1 andthe 5′ terminus −1181, there were specifically constructed the p1181plasmid having the sequence −1181 to −1 of the cucumisin promoter, thep865 plasmid having the sequence −865 to −1, the p371 plasmid having thesequence −371 to −1, the p310 plasmid having the sequence −310 to −1,the p254 plasmid having the sequence −254 to −1, the p234 plasmid havingthe sequence −234 to −1, the p214 plasmid having the sequence −214 to−1, the p200 plasmid having the sequence −200 to −1, the p89 plasmidhaving the sequence −89 to −1, the pKGX plasmid resulting from joiningthe −90 to −1 region of the cauliflower mosaic virus (CaMV) 35S promoterto the downstream portion of the sequence −254 to −215, and the pXplasmid having the −90 to −1 region of the cauliflower mosaic virus(CaMV) 35S promoter. pBI221 having an about 800 bp portion of thecauliflower mosaic virus (CaMV) 35S promoter was used as a positivecontrol (FIG. 3 to FIG. 6).

EXAMPLE 4 Site-Specific Expression of the Cucumisin Promoter

(1) DNA Shooting into Organs by the Particle Bombardment

An immature muskmelon fruit (about 4.5 cm in diameter) cut to piecesabout 1 to 2 mm in thickness and leaves and the stem cut to anappropriate size were placed in plastic dishes having a diameter of 9cm.

70% Ethanol (1 ml) was added to 60 mg of gold particles having adiameter of 1.6 μm, and the mixture was vortexed for 5 minutes, allowedto stand for 15 minutes and then centrifuged for 5 seconds, and thesupernatant was removed. Further, the procedure comprising addition of 1ml of sterilized water, 1 minute of vortexing, 1 minute of standing and2 seconds of centrifugation was repeated 3 times and, then, 50% glycerolwas added to make the final gold particle concentration 60 mg/ml.

While vigorously vortexing 50 μl of the gold particle preparation, 5 μl(containing about 1.2 pmole) of each solution of the p1181, p865, p371,p200, pSKGUS3C or pBI221 plasmid was added and, after further additionof 50 μl of a 2.5 M calcium chloride solution and 20 μl of a 0.1 Mspermidine solution, the mixture was vigorously vortexed for 3 minutes.After 1 minute of standing and 2 seconds of centrifugation, thesupernatant was removed and the pellet was resuspended in ethanol. Thepellet was placed on the middle of a microcarrier and dried.

The gold particles with each fusion gene adsorbed thereon were shottwice into samples of the immature fruit, leaves and stem for each kindof DNA. For the shooting, the Biolistic PDS-1000/He particle DeliverySystem (product of Bio-Rad) was used.

(2) GUS Activity Staining in Organs

After completion of the shooting, the dishes were closely sealed andwrapped with an aluminum foil for shielding against light, and incubatedat 25° C. for 24 hours. Each sample was further immersed in a GUSstaining solution (50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mMpotassium hexacyanoferrate(II), 0.5 mM potassium hexacyanoferrate(III),1 mM X-Gluc, 2% DMSO, 0.1% Triton X-100), tightly sealed, incubatedovernight in the dark at 37° C., then decolorized in ethanol, andchecked, by staining, for the expression of the GUS protein in theimmature melon fruit, mature leaf and stem by various cucumisinpromoters. The results are shown in Table 1.

TABLE 1 Plasmid Fruit Mature leaf Stem p1181 +++ − − p865 +++ − − p371+++ − − p200 − +/− +/− pSKGUS3C − − − pBI221 +++ +++ +++ +++: strongstaining +/−: very weak staining −: no staining

EXAMPLE 5 Identification of a Fruit-Specific Expression Regulating DNARegion in the Cucumisin Promoter

(1) Plasmids Used

The method of DNA shooting into various organs by the particlebombardment was the same as in Example 4, and the structures of theplasmids used were as shown in Example 3.

(2) Preparation of Extracts

The immature melon fruit and mature leaf samples prepared by the methodshown in Example 4 (1) were each ground in liquid nitrogen and, afterthawing at room temperature, 1× PicaGene cultured cell lysing agentcontaining 2 mM DFP was added, followed by grinding and centrifugation.Each supernatant was used as an extract sample.

(3) GUS Activity Assay

For determining the GUS activity, p35S-INT-LUC+(FIG. 7) produced byinserting, into the pUC19 vector, the cauliflower mosaic virus (CaMV)35S promoter, INT (first intron of the rice SOD gene) and LUC (fireflyluciferase gene) was shot simultaneously, and the luciferase activitythus assayed was used for GUS activity standardization.

For GUS activity assaying, 4-methylumbelliferyl-β-D-glucuronide wasadded to a final concentration of 1 mM to each extract prepared asdescribed above under (2), and the mixture was incubated at 37° C. for30 minutes. Further, 4 times volume of a 0.2 M sodium carbonate solutionwas added, and the fluorescence intensity was measured at the excitationwavelength 365 nm and measurement wavelength 455 nm using an F-3010fluorescence spectrophotometer (product of Hitachi). Separately, aregression line was prepared using 4-methylumbelliferone, and thefluorescence intensity measured was converted to the GUS activity (pmole4-MU/min).

For luciferase activity assaying, the PicaGene emission reagent of thePicaGene emission kit PGL 100 (product of Toyo Ink Manufacturing) wasadded to the extract prepared as described under (2), the mixture wasallowed to stand at room temperature for 30 seconds, and the emissionwas measured using a liquid scintillation counter (LSC-5100, product ofAloka). Separately, a regression line was prepared using the fireflyluciferase enzyme solution attached to the kit, and the emissionmeasured was converted to the luciferase enzyme amount (g), which wasrecorded as the luciferase activity.

The GUS activity obtained was divided by the luciferase activity forstandardization. For each of the melon fruit and leaf samples, the meanvalue and standard deviation was calculated from three standardized GUSactivity values for each fusion gene. Further, that mean value wasexpressed in terms of percentage relative to the mean standardized GUSactivity value obtained by shooting the positive control pBI221, and theresulting percentage was reported as the relative GUS activity. Theresults thus obtained are shown in Table 2.

TABLE 2 Plasmid Relative GUS activity introduced Fruit Mature leaf StempBI221 100 100 100 p1181 36 3 8 p310 42 1 3 p254 19 1 2 p234 39 1 4 p2146 1 1 p89 5 1 1 pSKGUS3C 0 0 0 * Shown as relative values with the GUSactivity at each site as obtained by using pBI221 being taken as 100.

EXAMPLE 6 Fruit-Specific Expression Using a Heterologous Promoter(Cauliflower Mosaic Virus (CaMV) 35S Promoter)

(1) Plasmids Used

DNA shooting into various organs by the particle gun method was carriedout in the same manner as in Example 4, and the structures of theplasmids were as shown in Example 3.

(2) Extract Preparation

The immature melon fruit and mature leaf samples prepared by the methodshown in Example 4 (1) were each ground in liquid nitrogen and, afterthawing at room temperature, 1× PicaGene cultured cell lysing agentcontaining 2 mM DFP was added, followed by grinding and centrifugation.Each supernatant was used as an extract sample.

(3) GUS Activity Assay

For the cauliflower mosaic virus (CaMV) 35S promoter with thefruit-specific expression regulating DNA sequence introduced therein,the relative GUS activities in melon fruit samples were measured by themethod according to Example 5. The results are shown in Table 3.

TABLE 3 Plasmid introduced GUS activity pBI221 100 pKGX 78 pX 15 p89 5 *Shown as relative values with the GUS activity obtained by using pBI221being taken as 100.

EXAMPLE 7 Identification, by a Gel Shift Assay, of the SequencePositively Acting on the Specific Expression and of the SequenceNegatively Acting Thereon

(1) Nucleoprotein Extraction from Prince Melon

An immature prince melon fruit was sliced, the seeds were removed, andthe core portion alone was taken out using a knife and ice-cooled (about320 g). Leaves were used after cutting to an appropriate size. Thesample was ground in a mixer with 500 ml of ice-cooled nucleusdisruption buffer (1 M hexylene glycol, 10 mM PIPES/KOH (pH 7.0), 10 mMMgCl₂, 5 mM 2-mercaptoethanol, 2 mM DFP, 0.2% Triton X-100) added. Theresulting mixture was filtered through a 50-μm nylon mesh, and thefiltrate was centrifuged at 2,000×g at 4° C. for 10 minutes. The pelletthus obtained was suspended in 80 ml of ice-cooled nucleus washingbuffer (0.5 M hexylene glycol, 10 mM PIPES/KOH (pH 7.0), 10 mM MgCl₂, 5mM 2-mercaptoethanol, 1 mM DFP, 0.2% Triton X-100), and a pellet wasrecovered by 5 minutes of centrifugation at 3,000×g at 4° C. The pelletwas suspended in 20 ml of ice-cooled nucleus dissolution buffer (110 mMKCl, 15 mM Hepes/KOH (pH 7.5), 5 mM MgCl₂, 5 mM 2-mercaptoethanol, 1 mMDTT, 5 μg/ml antipain, 5 μg/ml leupeptin, 5 μg/ml chymostatin), and 2 mlof a 4 M ammonium sulfate solution was added dropwise to the suspensionwith stirring. After 30 minutes of gentle shake culture, the culture wascentrifuged at 100,000×g at 4° C. for 90 minutes using a Beckman W/Ti 60rotor. Ammonium sulfate in a fine granular form, in an amount of 0.25 gper ml of the supernatant, was added portionwise and dissolved in thesupernatant. The mixture was allowed to stand overnight at 0° C. andthen centrifuged at 10,000×g at 4° C. for 15 minutes. The thus-obtainedpellet was suspended in 0.5 ml of ice-cooled nucleus extraction buffer(70 mM KCl, 25 mM Hepes/KOH (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 10%glycerol, 5 μg/ml antipain, 5 μg/ml leupeptin, 5 μg/ml chymostatin) anddialyzed against dialysis buffer (70 mM KCl, 25 mM Hepes/KOH (pH 7.5), 1mM mercaptoethanol, 0.1 mM EDTA, 20% glycerol) for 2 hours using theBio-Tech dialysis cup MWCO 8000 (product of Bio-Tech), during which thebuffer was exchanged four times. After dialysis, the dialyzate wascentrifuged at 12,000×g at 4° C. for 10 minutes, and the supernatantobtained was divided into portions and immediately frozen in liquidnitrogen and stored at −80° C. until testing.

(2) Probes

The G probe (SEQ ID NO:7), H probe (SEQ ID NO:8), Ga probe (basesequence represented by the bases Nos. 1 to 20 in SEQ ID NO:7), Gb probe(SEQ ID NO:1) and G5 probe (base sequence represented by the bases Nos.27 to 40 in SEQ ID NO:7) were each terminally labeled with [γ-³²P]ATPusing T4 kinase.

(3) Gel Shift Assay

The binding reaction was carried out as follows. 3 μl of 5× bindingbuffer (200 mM KCl, 125 mM Hepes/KOH (pH 7.5), 5 mM mercaptoethanol, 0.5mM EDTA, 50% glycerol), 2 μl of 1 μg/μl poly(dI-dC)·(dI-dC) (product ofPharmacia), 300,000 cpm of the labeled probe (prepared as describedabove under (2)), 3 μl of the prince melon fruit core or leaf nucleusextract of 1 μg/μl concentration (prepared as described above under (1))were added to each well, the whole amount was made 15 μl usingsterilized water, and incubation was carried out at 25° C. for 15minutes. Each reaction mixture was electrophoresed on an undenatured 30%acrylamide gel at a constant current of 20 mA for 30 minutes, the gelwas then transferred to a filter paper and dried with a gel drier. An Xray film was exposed thereto at −80° C. for 3 hours and then developed.In the case of competitive gel shift assay, the unlabeled probe, in anamount 100 times that of the labeled probe, was added as a competitiveprobe and the binding reaction was carried out. The results are shown inTable 4.

TABLE 4 Probe Fruit Leaf Probe G 100 0 Probe G + unlabeled probe G 0 0Probe H 5 0 * Shown in terms of relative values with the specific bandfor the probe G being taken as 100.

As a result, fruit-specific strong nucleoprotein binding was observedwith the G probe. Fruit-specific nucleoprotein binding was also foundwith the labeled Ga probe and labeled Gb probe. The GUS activity datasuggest that a nucleoprotein negatively acting on the gene expressionbind to the Ga probe portion and one positively acting thereon to the Gbprobe moiety.

When the Gb probe labeled in the same manner was used and subjected tocompetition with the unlabeled G5 probe, the fruit-specific banddisappeared almost completely. On the other hand, when probes derivedfrom the G5 probe by mutation of one base were subjected to competitionas unlabeled probes, the disappearance of the specific band becameslight with the G5 probe-derived probe resulting from mutation of thethird base T to C and the one resulting from mutation of the 7th base Ato G. This indicates that the bases in those portions constitutesequences more important for the binding of the transcription factorsupposedly involved in the fruit-specific expression.

INDUSTRIAL APPLICABILITY

By utilizing the DNA sequence of the invention, it has become possibleto cause specific expression of a desired protein in plant fruit. As aresult, the isolation and purification of a useful protein or usefulsubstance can be facilitated by harvesting such fruit.

1. A fruit-specific expression-regulating unit DNA sequence consistingof the DNA sequence −1181 to −1 of a cucumisin promoter that consists ofthe base sequence under SEQ ID NO:3 when the 3′ terminus of said basesequence is numbered −1 and the 5′ terminus thereof is numbered −1181.2. A fruit-specific expression-regulating unit DNA sequence consistingof the DNA sequence −865 to −1 of a cucumisin promoter that consists ofthe base sequence under SEQ ID NO:3 when the 3′ terminus of said basesequence is numbered −1 and the 5′ terminus thereof is numbered −1181.3. A fruit-specific expression-regulating unit DNA sequence consistingof the DNA sequence −371 to −1 of a cucumisin promoter that consists ofthe base sequence under SEQ ID NO:3 when the 3′ terminus of said basesequence is numbered −1 and the 5′ terminus thereof is numbered −1181.4. A fruit-specific expression-regulating unit DNA sequence consistingof the DNA sequence −310 to −1 of a cucumisin promoter that consists ofthe base sequence under SEQ ID NO:3 when the 3′ terminus of said basesequence is numbered −1 and the 5′ terminus thereof is numbered −1181.5. A fruit-specific expression-regulating unit DNA sequence consistingof the DNA sequence −254 to −1 of a cucumisin promoter that consists ofthe base sequence under SEQ ID NO:3 when the 3′ terminus of said basesequence is numbered −1 and the 5′ terminus thereof is numbered −1181.6. A fruit-specific expression-regulating unit DNA sequence consistingof the DNA sequence −234 to −1 of a cucumisin promoter that consists ofthe base sequence under SEQ ID NO:3 when the 3′ terminus of said basesequence is numbered −1 and the 5′ terminus thereof is numbered −1181.7. A plasmid which comprises a DNA coding for a structural gene productthat is expressed in a plant or for a corresponding antisense RNA, aterminator for said DNA, which terminator functions in the plant, andthe DNA sequence according to claim 1 for regulating specific expressionof said DNA in said plant.
 8. A plasmid which comprises a DNA coding fora structural gene product that is expressed in a plant or for acorresponding antisense RNA, a terminator for said DNA, which terminatorfunctions in the plant, and the DNA sequence according to claim 2 forregulating specific expression of said DNA in said plant.
 9. A plasmidwhich comprises a DNA coding for a structural gene product that isexpressed in a plant or for a corresponding antisense RNA, a terminatorfor said DNA, which terminator functions in the plant, and the DNAsequence according to claim 3 for regulating specific expression of saidDNA in said plant.
 10. A plasmid which comprises a DNA coding for astructural gene product that is expressed in a plant or for acorresponding antisense RNA, a terminator for said DNA, which terminatorfunctions in the plant, and the DNA sequence according to claim 4 forregulating specific expression of said DNA in said plant.
 11. A plasmidwhich comprises a DNA coding for a structural gene product that isexpressed in a plant or for a corresponding antisense RNA, a terminatorfor said DNA, which terminator functions in the plant, and the DNAsequence according to claim 5 for regulating specific expression of saidDNA in said plant.
 12. A plasmid which comprises a DNA coding for astructural gene product that is expressed in a plant or for acorresponding antisense RNA, a terminator for said DNA, which terminatorfunctions in the plant, and the DNA sequence according to claim 6 forregulating specific expression of said DNA in said plant.
 13. A plantcell which is transformed with the plasmid according to claim
 7. 14. Aplant cell which is transformed with the plasmid according to claim 8.15. A plant cell which is transformed with the plasmid according toclaim
 9. 16. A plant cell which is transformed with the plasmidaccording to claim
 10. 17. A plant cell which is transformed with theplasmid according to claim
 11. 18. A plant cell which is transformedwith the plasmid according to claim
 12. 19. A plant body which istransformed with the plasmid according to claim
 7. 20. A plant bodywhich is transformed with the plasmid according to claim
 8. 21. A plantbody which is transformed with the plasmid according to claim
 9. 22. Aplant body which is transformed with the plasmid according to claim 10.23. A plant body which is transformed with the plasmid according toclaim
 11. 24. A plant body which is transformed with the plasmidaccording to claim
 12. 25. A microorganism harboring the plasmidaccording to claim
 7. 26. A microorganism harboring the plasmidaccording to claim
 8. 27. A microorganism harboring the plasmidaccording to claim
 9. 28. A microorganism harboring the plasmidaccording to claim
 10. 29. A microorganism harboring the plasmidaccording to claim
 11. 30. A microorganism harboring the plasmidaccording to claim 12.